Compositions and methods for reducing bioburden in chromatography

ABSTRACT

The invention provides methods for microbial bioburden reduction of various chromatography matrices, including bioburden reduction in the context of large-scale Protein A-based affinity chromatography columns.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefits of U.S. Provisional PatentApplication No. 62/452,140, filed on Jan. 30, 2017, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides methods for microbial bioburden reductionof various chromatography matrices, including bioburden reduction in thecontext of large-scale Protein A-based affinity chromatography columns.

BACKGROUND OF THE INVENTION

Antibody drugs are the most prevalent biopharmaceutical products.Affinity chromatography, e.g., performed with a natural or engineeredstaphylococcal protein A ligand, is widely used as a capture method inthe antibody drug manufacturing process to remove impurities andcontaminants. Protein A binds the Fc-region of antibodies, with proteinA columns considered to be selective for purification of monoclonalantibodies. Affinity chromatography with protein A typically involves aclean-in-place (CIP) step to clean and remove impurities that are boundto the column, such as precipitated or denatured substances. CIP isnormally performed with a sodium hydroxide solution.

In addition to cleaning, sodium hydroxide solutions, or phosphoric acidsolutions with benzyl alcohol, are used to reduce the number of microbesin a protein A chromatography matrix or column. Bacteria from the mediaused to culture monoclonal-antibody producing cells, as well asassociated host cell proteins and DNA, can quickly increase thebioburden of a protein A column during use. The bioburden increases assuch bacteria and microbes accumulate on the column. Column performancegenerally degrades as bioburden increases. Signs of such degradationinclude decrease in product purity, column packing deterioration, andincreased backpressure.

Managing and reducing microbial bioburden on protein A columns isimportant because protein A columns are very expensive, with the packingand unpacking of such affinity columns being labor intensive. To avoidexpenses in replacing the protein A column or adding process stepsupstream of protein A column purification, there is a need to findagents that remove a significant amount of bioburden from the columnquickly without negatively affecting the structure and function ofprotein A, and that have few downstream effects.

The importance of microbial bioburden reduction is not limited toprotein A chromatography matrices but encompasses other chromatographymatrices with proteinaceous ligands coupled to a support as well asmatrices not involving proteinaceous ligands such as, e.g., various ionexchange chromatography matrices, hydrophobic interaction chromatography(HIC) matrices, mixed mode chromatography matrices, size exclusionchromatography matrices, etc.

Microbial bioburden reduction of chromatography matrices is particularlyimportant in the context of a good manufacturing practice (GMP), orcurrent good manufacturing practice (CGMP). Such practices must provideconsistency in manufacturing steps and quality of product so as to meetrequirements of regulatory bodies, such as the U.S. Food and DrugAdministration. GMP and CGMP require a high degree of predictability andstandardization in manufacturing processes, particularly with ensuringpurity of the manufactured therapeutic biomolecules used in humanpatients. With the labor involved in growing cultures to producebiomolecules, great expense arises when a failure occurs. An excessivebioburden can decrease column performance, which can interfere withpurifying the product in a standardized and predictable way and possiblycause other failure points to be triggered.

The agents presently known in the art to reduce bioburden have negativedownstream effects. For example, solutions based on sodium hydroxide,and those based on phosphoric acid with benzyl alcohol, may be effectiveto kill microorganisms but also tend to denature the proteinaceiousligands (e.g., protein A) thus negatively affecting their function.

Further, oxidants and other components of such solutions can remain withthe purified monoclonal antibodies and further degrade them downstream.

Thus, those of ordinary skill in the art appreciate that it is difficultto reduce bioburden in chromatography matrices, especially in matriceswith proteinaceous ligands, such as protein A chromatography matrices.

SUMMARY OF THE INVENTION

As specified above, there is a need for new effective methods that canbe used in the context of large-scale GMP and CGMP to reduce microbialbioburden of various chromatography matrices, and especially thoseinvolving proteinaceous ligands such as protein A. The present inventionaddresses this and other needs by providing compositions and methods formicrobial bioburden reduction of chromatography matrices.

In one aspect, the present invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 0.1 Mto about 0.5 M acetic acid, wherein the contacting step is performed forat least about 2 hours.

In a related aspect, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 0.5 Mto about 1.0 M acetic acid, wherein the contacting step is performed forat least about 1 hour.

In a related aspect, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 0.1 Mto about 1.0 M acetic acid, wherein the contacting step results in oneor more of a reduction in the amount of spore forming bacteria by atleast 3 log₁₀, a reduction in the amount of gram positive bacteria by atleast 5 log₁₀, and a reduction in the amount of gram negative bacteriaby at least 5 log₁₀ in the chromatography matrix.

In a separate aspect, the present invention provides a method formicrobial bioburden reduction of a chromatography matrix, comprisingcontacting the chromatography matrix with a composition comprising fromabout 4.0 M to about 12.0 M urea, wherein the contacting step isperformed for at least about 30 minutes.

In a related aspect, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 4.0 Mto about 12.0 M urea, wherein the contacting step results in one or moreof a reduction in the amount of spore forming bacteria by at least 2log₁₀, a reduction in the amount of gram positive bacteria by at least 5log₁₀, and a reduction in the amount of gram negative bacteria by atleast 5 log₁₀, in the chromatography matrix.

In one embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 0.5 M acetic acid, wherein the contacting step isperformed for at least about 4 hours.

In another embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 0.1 M acetic acid and about 20% ethanol, whereinthe contacting step is performed for at least about 4 hours.

In a further embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 8 M urea, wherein the contacting step is performedfor at least about 1 hour.

In yet another embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 8 M urea and about 20% ethanol, wherein thecontacting step is performed for at least about 1 hour.

In one embodiment, the invention provides a method for reducingmicrobial load before applying a composition comprising a pharmaceuticalagent for purification comprising (a) providing a chromatography matrix;(b) performing any of the above methods of the invention; and (c)applying the composition comprising the pharmaceutical agent to thechromatography matrix.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in the following description, claimsand drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of a spike study in solution with 0.5 Macetic acid. The extent of bacterial killing is measured in solution,without chromatography matrix present. The black bars represent theamount of Bacillus pseudofirmus and the diagonally striped barsrepresent the amount of Microbacierium species in a MabSelect™ Xtracolumn before exposure with 0.5 acetic acid (T=0) and one hour (T=1 hr)after exposure with acetic acid.

FIG. 2 illustrates the results of killing Bacillus psuedofirmus insolution, without chromatography matrix present, by spiking the solutionwith Bacillus pseudofirmus and measuring the bacterial titer. Thefollowing agents were added to separate solutions: (a) water forinjection (WFI), (b) 8 M urea, (c) 8 M urea and 20% ethanol, (d) 6 Mguanidine hydrochloride, (e) guanidine hydrochloride with 20% ethanol. Aspike confirmation measurement in PBS was taken for each, as well asmeasurements at the 0 minute, 30 minute, and 60 minute time points. Theblack bar is for WFI, the horizontally striped bar is for 8 M urea, thewhite bar is for 8 M urea and 20% ethanol, the diagonally striped bar isfor 6 M guanidine hydrochloride and the cross-hatched bar is for 6 Mguanidine hydrochloride and 20% ethanol.

FIG. 3 illustrates the results of killing Microbacterium species insolution, without chromatography matrix present, by spiking the solutionwith Bacillus pseudofirmus and measuring the bacterial titer. Thefollowing agents were added: (a) water for injection (WFI), (b) 8 Murea, (c) 8 M urea and 20% ethanol, (d) 6 M guanidine hydrochloride, (e)guanidine hydrochloride with 20% ethanol. A spike confirmationmeasurement in PBS was taken, as well as measurements at the 0 minute,30 minute, and 60 minute time points. The black bar is for WFI, thehorizontally striped bar is for 8 M urea, the white bar is for 8 M ureaand 20% ethanol, the diagonally striped bar is for 6 M guanidinehydrochloride and the cross-hatched bar is for 6 M guanidinehydrochloride and 20% ethanol.

FIG. 4 illustrates the results of killing Stenotrophomonas maltophiliain solution, without chromatography matrix present, by spiking thesolution with Bacillus pseudofirmus and measuring the bacterial titer.The following agents were added: (a) water for injection (WFI), (b) 8 Murea, (c) 8 M urea and 20% ethanol, (d) 6 M guanidine hydrochloride, (e)guanidine hydrochloride with 20% ethanol. A spike confirmationmeasurement in PBS was taken, as well as measurements at the 0 minute,30 minute, and 60 minute time points. The black bar is for WFI, thehorizontally striped bar is for 8 M urea, the white bar is for 8 M ureaand 20% ethanol, the diagonally striped bar is for 6 M guanidinehydrochloride and the cross-hatched bar is for 6 M guanidinehydrochloride and 20% ethanol.

FIGS. 5-9 illustrate the results of various product quality studiesperformed on Protein A-containing resins (MabSelect™ Xtra and MabSelect™SuRe) exposed to 0.5 M acetic acid for different lengths of time. Thefilled circles show the values for MabSelect™ Xtra that was exposed to0.5 M acetic acid for 375 hours, or not exposed at all. The cross marksshow the values for MabSelect™ SuRe that was not exposed to 0.5 M aceticacid for 5, 10, 25, 200 or 400 hours, or not exposed at all.

FIGS. 10-14 illustrate an ANOVA analysis of various product qualitystudies performed on the MabSelect™ Xtra Protein A resin. In thepost-acid column, the filled circles reflect the values from Table 1 of375 hours of exposure to 0.5 M acetic acid. In the pre-acid column, thefilled circles reflect the values from Table 1 of zero hours of exposureto 0.5 M acetic acid. The diamond shapes indicate a range based on the95% confidence intervals. All of the ranges for post-acid overlap withthose of pre-acid. The ANOVA analysis shows no statistically significantnegative effect on protein quality from prolonged exposure of the resinto 0.5 M acetic acid.

FIGS. 15-19 illustrate an ANOVA analysis of various product qualitystudies performed on the MabSelect™ SuRe Protein A resin. In thepost-acid column, the filled circles reflect the values from Table 1 of400 hours of exposure to 0.5 M acetic acid. In the pre-acid column, thefilled circles reflect the values from Table 1 of zero hours of exposureto 0.5 M acetic acid. The diamond shapes indicate a range based on the95% confidence intervals. In FIG. 15, the range is greater afterexposure to acid to a statistically significant degree. In FIGS. 16-19,the ranges for pre-acid overlap with those for post-acid. The ANOVAanalysis shows no statistically significant negative effect on proteinquality from prolonged exposure of the resin to 0.5 M acetic acid.

DETAILED DESCRIPTION

In one aspect, the present invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 0.1 Mto about 0.5 M acetic acid, wherein the contacting step is performed forat least about 2 hours. In various embodiments, the contacting step isperformed for 2 to 5 hours, 2 to 10 hours, 2 to 25 hours, 2 to 200hours, 2 to 375 hours, or 2 to 400 hours. In one embodiment, thecontacting step is performed for at least about 4 hours. In variousembodiments, the contacting step is performed for 4 to 5 hours, 4 to 10hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours.In one embodiment, the composition comprises about 0.1 M acetic acid andthe contacting step is performed for at least about 4 hours. In oneembodiment, the composition comprises about 0.5 M acetic acid and thecontacting step is performed for at least about 4 hours. In variousembodiments, the composition comprises about 0.5 M acetic acid and thecontacting step is performed for 4 to 5 hours, 4 to 10 hours, 4 to 25hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours.

In one embodiment, the composition further comprises an alcohol.Non-limiting examples of alcohols that can be used include ethanol(e.g., about 20%) and benzyl alcohol (e.g., from about 1% to about 2%).In one specific embodiment, the composition consists essentially ofabout 0.1M acetic acid and about 20% ethanol.

In one embodiment, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition consisting essentially ofabout 0.1 M to about 0.5 M acetic acid, wherein the contacting step isperformed for at least about 2 hours. In one specific embodiment, thecontacting step is performed for at least about 4 hours. In variousembodiments, the contacting step is performed for 4 to 5 hours, 4 to 10hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours.In one specific embodiment, the composition consists essentially ofabout 0.1 M acetic acid and the contacting step is performed for atleast about 4 hours. In another specific embodiment, the compositionconsists essentially of about 0.5 M acetic acid and the contacting stepis performed for at least about 4 hours. In various embodiments, thecomposition comprises about 0.5 M acetic acid and the contacting step isperformed for 4 to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200hours, 4 to 375 hours, or 4 to 400 hours.

In a related aspect, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 0.5 Mto about 1.0 M acetic acid, wherein the contacting step is performed forat least about 1 hour. In various embodiments, the contacting step isperformed for 1 to 5 hours, 1 to 10 hours, 1 to 25 hours, 1 to 200hours, 1 to 375 hours, 1 to 400 hours, 4 to 5 hours, 4 to 10 hours, 4 to25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours. In oneembodiment, the composition comprises about 0.5 M acetic acid and thecontacting step is performed for at least about 1 hour. In variousembodiments, the composition comprises about 0.5 M acetic acid and thecontacting step is performed for 1 to 5 hours, 1 to 10 hours, 1 to 25hours, 1 to 200 hours, 1 to 375 hours, 1 to 400 hours, 4 to 5 hours, 4to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400hours.

In one embodiment, the composition further comprises an alcohol.Non-limiting examples of alcohols that can be used include ethanol(e.g., about 20%) and benzyl alcohol (e.g., from about 1% to about 2%).In one specific embodiment, the composition consists essentially ofabout 0.5 M acetic acid and about 20% ethanol.

In one embodiment, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition consisting essentially ofabout 0.5 M to about 1.0 M acetic acid, wherein the contacting step isperformed for at least about 1 hour. In various embodiments, thecomposition consists essentially of about 0.5 M to about 1.0 M aceticacid and the contacting step is performed for 1 to 5 hours, 1 to 10hours, 1 to 25 hours, 1 to 200 hours, 1 to 375 hours, 1 to 400 hours, 4to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375hours, or 4 to 400 hours. In one specific embodiment, the compositionconsists essentially of about 0.5 M acetic acid and the contacting stepis performed for at least about 1 hour. In various embodiments, thecomposition consists essentially of about 0.5 M acetic acid and thecontacting step is performed for 1 to 5 hours, 1 to 10 hours, 1 to 25hours, 1 to 200 hours, 1 to 375 hours, 1 to 400 hours, 4 to 5 hours, 4to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400hours.

In a related aspect, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 0.1 Mto about 1.0 M acetic acid, wherein the contacting step results in oneor more of a reduction in the amount of spore forming bacteria (e.g.,Bacillus pseudofirmus) by at least 3 log₁₀, a reduction in the amount ofgram positive bacteria (e.g., Microbacterium spp.) by at least 5 log₁₀,and a reduction in the amount of gram negative bacteria (e.g.,Stenotrophomonas maltophilia) by at least 5 log₁₀ in the chromatographymatrix. In one specific embodiment, the contacting step results in areduction in the amount of one or more of spore forming bacteria (e.g.,Bacillus pseudofirmus), gram positive bacteria (e.g., Microbacteriumspp.), and gram negative bacteria (e.g., Stenotrophomonas maltophilia),in the chromatography matrix, to below the limit of detection asdetermined by an assay, such as, for example, (1) a biofiltration assay,(2) microscopic bacterial staining, (3) IR/FTIR spectroscopy method, (4)a sterility test, or (5) a bacterial identification test. In variousembodiments, the contacting step is performed for at least about 1 hour,1 to 5 hours, 1 to 10 hours, 1 to 25 hours, 1 to 200 hours, 1 to 375hours, 1 to 400 hours, for at least about 4 hours, for 4 to 5 hours, 4to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400hours.

In one embodiment, the composition further comprises an alcohol.Non-limiting examples of alcohols that can be used include ethanol(e.g., about 20%) and benzyl alcohol (e.g., from about 1% to about 2%).In one specific embodiment, the composition consists essentially ofabout 0.1 M acetic acid and about 20% ethanol. In one specificembodiment, the composition consists essentially of about 0.5 M aceticacid and about 20% ethanol.

In one embodiment, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition consisting essentially ofabout 0.1 M to about 1.0 M acetic acid, wherein the contacting stepresults in one or more of a reduction in the amount of spore formingbacteria (e.g., Bacillus pseudofirmus) by at least 3 log₁₀, a reductionin the amount of gram positive bacteria (e.g., Microbacterium spp.) byat least 5 log₁₀, and a reduction in the amount of gram negativebacteria (e.g., Stenotrophomonas maltophilia) by at least 5 log₁₀, inthe chromatography matrix. In one specific embodiment, the contactingstep results in a reduction in the amount of one or more of sporeforming bacteria (e.g., Bacillus pseudofirmus), gram positive bacteria(e.g., Microbacterium spp.), and gram negative bacteria (e.g.,Stenotrophomonas maltophilia), in the chromatography matrix, to belowthe limit of detection as determined by an assay, such as, for example,(1) a biofiltration assay, (2) microscopic bacterial staining, (3)IR/FTIR spectroscopy method, (4) a sterility test, or (5) a bacterialidentification test. In various embodiments, the contacting step isperformed for at least about 1 hour, 1 to 5 hours, 1 to 10 hours, 1 to25 hours, 1 to 200 hours, 1 to 375 hours, 1 to 400 hours, at least about4 hours, 4 to 5 hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4to 375 hours, or 4 to 400 hours.

In one embodiment of any of the above methods of the invention, thecomposition further comprises an acetate salt.

In one embodiment of any of the above methods of the invention, thecomposition has pH between about 2 and about 3.

In a separate aspect, the present invention provides a method formicrobial bioburden reduction of a chromatography matrix, comprisingcontacting the chromatography matrix with a composition comprising fromabout 4.0 M to about 12.0 M urea, wherein the contacting step isperformed for at least about 30 minutes. In one embodiment, thecontacting step is performed for at least about 1 hour. In oneembodiment, the composition comprises about 8 M urea.

In one embodiment, the composition further comprises an alcohol.Non-limiting examples of alcohols that can be used include ethanol(e.g., about 20%) and benzyl alcohol (e.g., from about 1% to about 2%).In one specific embodiment, the composition consists essentially ofabout 8 M urea and about 20% ethanol.

In one embodiment, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition consisting essentially ofabout 4.0 M to about 12.0 M urea, wherein the contacting step isperformed for at least about 30 minutes. In one specific embodiment, thecontacting step is performed for at least about 1 hour. In one specificembodiment, the composition consists essentially of 8 M urea.

In a related aspect, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 4.0 Mto about 12.0 M urea, wherein the contacting step results in one or moreof a reduction in the amount of spore forming bacteria (e.g., Bacilluspseudofirmus) by at least 2 log₁₀, a reduction in the amount of grampositive bacteria (e.g., Microbacterium spp.) by at least 5 log₁₀, and areduction in the amount of gram negative bacteria (e.g.,Stenotrophomonas maltophilia) by at least 5 log₁₀, in the chromatographymatrix. In one specific embodiment, the contacting step results in areduction in the amount of one or more of spore forming bacteria (e.g.,Bacillus pseudofirmus), gram positive bacteria (e.g., Microbacteriumspp.), and gram negative bacteria (e.g., Stenotrophomonas maltophilia),in the chromatography matrix, to below the limit of detection asdetermined by an assay, such as, for example, (1) a biofiltration assay,(2) microscopic bacterial staining, (3) IR/FTIR spectroscopy method, (4)a sterility test, or (5) a bacterial identification test.

In one embodiment, the composition further comprises an alcohol.Non-limiting examples of alcohols that can be used include ethanol(e.g., about 20%) and benzyl alcohol (e.g., from about 1% to about 2%).In one specific embodiment, the composition consists essentially ofabout 8 M urea and about 20% ethanol.

In one embodiment, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition consisting essentially ofabout 4.0 M to about 12.0 M urea, wherein the contacting step results inone or more of a reduction in the amount of spore forming bacteria(e.g., Bacillus pseudofirmus) by at least 2 log₁₀, a reduction in theamount of gram positive bacteria (e.g., Microbacterium spp.) by at least5 log₁₀, and a reduction in the amount of gram negative bacteria (e.g.,Stenotrophomonas maltophilia) by at least 5 log₁₀, in the chromatographymatrix. In one specific embodiment, the contacting step results in areduction in the amount of one or more of spore forming bacteria (e.g.,Bacillus pseudofirmus), gram positive bacteria (e.g., Microbacteriumspp.), and gram negative bacteria (e.g., Stenotrophomonas maltophilia),in the chromatography matrix, to below the limit of detection asdetermined by an assay, such as, for example, (1) a biofiltration assay,(2) microscopic bacterial staining, (3) IR/FTIR spectroscopy method, (4)a sterility test, or (5) a bacterial identification test.

In a further aspect, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 4.0 Mto about 12.0 M guanidine hydrochloride, wherein the contacting step isperformed for at least about 30 minutes. In one embodiment, thecontacting step is performed for at least about 1 hour. In oneembodiment, the composition comprises about 6 M guanidine hydrochloride.

In one embodiment, the composition further comprises an alcohol.Non-limiting examples of alcohols that can be used include ethanol(e.g., about 20%) and benzyl alcohol (e.g., from about 1% to about 2%).In one specific embodiment, the composition consists essentially ofabout 6 M guanidine hydrochloride and about 20% ethanol.

In one embodiment, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition consisting essentially ofabout 4.0 M to about 12.0 M guanidine hydrochloride, wherein thecontacting step is performed for at least about 30 minutes. In onespecific embodiment, the contacting step is performed for at least about1 hour. In one specific embodiment, the composition consists essentiallyof about 6 M guanidine hydrochloride.

In a related aspect, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition comprising from about 4.0 Mto about 12.0 M guanidine hydrochloride, wherein the contacting stepresults in one or more of a reduction in the amount of spore formingbacteria (e.g., Bacillus pseudofirmus) by at least 2 log₁₀, a reductionin the amount of gram positive bacteria (e.g., Microbacterium spp.) byat least 4 log₁₀, and a reduction in the amount of gram negativebacteria (e.g., Stenotrophomonas maltophilia) by at least 2 log₁₀, inthe chromatography matrix. In one specific embodiment, the contactingstep results in a reduction in the amount of one or more of sporeforming bacteria (e.g., Bacillus pseudofirmus), gram positive bacteria(e.g., Microbacterium spp.), and gram negative bacteria (e.g.,Stenotrophomonas maltophilia), in the chromatography matrix, to belowthe limit of detection as determined by an assay, such as, for example,(1) a biofiltration assay, (2) microscopic bacterial staining, (3)IR/FTIR spectroscopy method, (4) a sterility test, or (5) a bacterialidentification test.

In one embodiment, the composition further comprises an alcohol.Non-limiting examples of alcohols that can be used include ethanol(e.g., about 20%) and benzyl alcohol (e.g., from about 1% to about 2%).In one specific embodiment, the composition consists essentially ofabout 6 M guanidine hydrochloride and about 20% ethanol.

In one embodiment, the invention provides a method for microbialbioburden reduction of a chromatography matrix, comprising contactingthe chromatography matrix with a composition consisting essentially ofabout 4.0 M to about 12.0 M guanidine hydrochloride, wherein thecontacting step results in one or more of a reduction in the amount ofspore forming bacteria (e.g., Bacillus pseudofirmus) by at least 2log₁₀, a reduction in the amount of gram positive bacteria (e.g.,Microbacterium spp.) by at least 4 log₁₀, and a reduction in the amountof gram negative bacteria (e.g., Stenotrophomonas maltophilia) by atleast 2 log₁₀, in the chromatography matrix. In one specific embodiment,the contacting step results in a reduction in the amount of one or moreof spore forming bacteria (e.g., Bacillus pseudofirmus), gram positivebacteria (e.g., Microbacterium spp.), and gram negative bacteria (e.g.,Stenotrophomonas maltophilia), in the chromatography matrix, to belowthe limit of detection as determined by an assay, such as, for example,(1) a biofiltration assay, (2) microscopic bacterial staining, (3)IR/FTIR spectroscopy method, (4) a sterility test, or (5) a bacterialidentification test.

In one aspect, the invention provides a method for microbial bioburdenreduction of a chromatography matrix, comprising contacting thechromatography matrix with a composition comprising from about 0.5 M toabout 1.0 M acetic acid and (i) from about 4.0 M to about 12.0 M ureaand/or (ii) from about 4.0 M to about 12.0 M guanidine hydrochloride,wherein the contacting step is performed for at least about 1 hour. Inone specific embodiment, the composition further comprises an alcohol.Non-limiting examples of alcohols that can be used include ethanol(e.g., about 20%) and benzyl alcohol (e.g., from about 1% to about 2%).

In one embodiment of any of the above methods of the invention, thecontacting step is conducted at a temperature between 15° C. and 30° C.In one specific embodiment, the contacting step is conducted at atemperature between 20° C. and 25° C.

In one embodiment of any of the above methods of the invention, thecomposition is substantially free of oxidants.

In one embodiment of any of the above methods of the invention, thecomposition does not comprise a peroxyacid.

In one embodiment of any of the above methods of the invention, thecomposition does not comprise a peroxide.

In one embodiment of any of the above methods of the invention, thecomposition does not comprise NaOH.

In one embodiment of any of the above methods of the invention, thecontacting step is repeated at least once.

In one embodiment of any of the above methods of the invention, thechromatography matrix is packed in a chromatography column. In onespecific embodiment, the chromatography column has an inner diameterbetween 0.5 cm and 1.5 cm and a bed height between 15 cm and 30 cm. Inone specific embodiment, the chromatography column has an inner diameterof about 1 cm and a bed height of about 20 cm. In one specificembodiment, the chromatography column has an inner diameter between 40cm and 1.6 meters and a bed height between 15 cm and 30 cm. In onespecific embodiment, the chromatography column has an inner diameter ofabout 1.4 meters and a bed height of about 20 cm.

In one embodiment of any of the above methods of the invention, thechromatography matrix comprises a proteinaceous ligand coupled to asupport. In one specific embodiment, the proteinaceous ligand comprisesone or more immunoglobulin binding domains. In one specific embodiment,the proteinaceous ligand is Protein A or a fragment or a derivativethereof. In one specific embodiment, the proteinaceous ligand isselected from the group consisting of Staphylococcus Protein A,Peptostreptococcus Protein L, Streptococcus Protein G, StreptococcusProtein A, and fragments and derivatives thereof. In one specificembodiment, the chromatography matrix is selected from the groupconsisting of MabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, MabSelect™SuRe pcc, MabSelect™ SuRe LX, MabCapture™ A, nProtein A Sepharose 4 FastFlow, Protein A Sepharose 4 Fast Flow, Protein A Mag Sepharose, ProteinA Sepharose CL-4B, rmp Protein A Sepharose Fast Flow, rProtein ASepharose 4 Fast Flow, Capto™ L, ProSep™-A, ProSep Ultra Plus,AbSolute™, CaptivA™ PriMab™, Protein A Diamond, Eshmuno™ A, Toyopearl™AF-rProtein A, Amsphere™ Protein A, KanCapA™, Protein G Mag SepharoseXtra, and Protein G Sepharose 4 Fast Flow. In one specific embodiment,the chromatography matrix is selected from the group consisting ofMabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, MabSelect™ SuRe PCC, andMabSelect™ SuRe LX. In one specific embodiment, the proteinaceous ligandis not measurably denatured after the method is performed.

In one embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 0.5 M acetic acid, wherein the contacting step isperformed for at least about 4 hours.

In another embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 0.1 M acetic acid and about 20% ethanol, whereinthe contacting step is performed for at least about 4 hours. In variousembodiments, the contacting step is performed for at least about 1 hour,1 to 5 hours, 1 to 10 hours, 1 to 25 hours, 1 to 200 hours, 1 to 375hours, 1 to 400 hours, at least about 4 hours, 4 to 5 hours, 4 to 10hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or 4 to 400 hours.

In a further embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 8 M urea, wherein the contacting step is performedfor at least about 1 hour.

In yet another embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 8 M urea and about 20% ethanol, wherein thecontacting step is performed for at least about 1 hour.

In a further embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 6 M guanidine hydrochloride, wherein the contactingstep is performed for at least about 1 hour.

In another embodiment, the invention provides a method for microbialbioburden reduction of MabSelect™ Xtra chromatography matrix, comprisingcontacting the chromatography matrix with a composition consistingessentially of about 6 M guanidine hydrochloride and about 20% ethanol,wherein the contacting step is performed for at least about 1 hour.

In one embodiment, the invention provides a method for reducingmicrobial load before applying a composition comprising a pharmaceuticalagent for purification comprising (a) providing a chromatography matrix;(b) performing any of the above methods of the invention; and (c)applying the composition comprising the pharmaceutical agent to thechromatography matrix.

It is to be understood that this invention is not limited to particularmethods and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention is defined by the claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The terms “about” and “approximately” are used interchangeably to meanwithin a statistically meaningful range of a value. Such a range can bewithin 50%, more preferably within 20%, still more preferably within10%, and even more preferably within 5% of a given value or range.

As used herein, the terms “microbe” or “microorganism” encompassprokaryotic organisms including bacteria and archaea, and eukaryoticorganisms, including fungi. These terms encompass both live cells andspores (for spore-forming organisms) as well as microbial products suchas, e.g., endotoxins.

The terms “microbial bioburden reduction” and “microbe bioburdenreduction” as used herein combines killing of microbes and someinterference with the interaction between a microbe and a chromatographymatrix. The reduction of microbial bioburden according to the presentinvention is not the same as any previously disclosed sanitizationprocess that was designed to kill essentially all, or even at least 99%,of the microbes that might exist on a chromatography column or matrix.

Rather, the present invention includes methods capable of killing lessthan 80%, less than 70%, less than 60%, or 40-60% of non-spore formingmicrobes. In such an embodiment, less than 50%, less than 40%, less than30%, less than 20% or less than 10%, or 10-20% of the spore formingmicrobes, such as Bacillus, on a column would be killed by the methodsof the present invention.

Even though the present invention includes methods that do not kill allof the bacteria on a column, at least 85%, at least 90%, at least 95%,at least 97%, about 98%, about 99% or about 100% of the viable microbescapable of being detected by the biofiltration assay, or any othermicrobiological assay disclosed herein and known in the art are removedfrom the chromatography matrix after treatment. In some embodiments ofthe present invention, the level of microbial bioburden is reduced belowGMP-acceptable levels in a pre-use flush sample, in an equilibrationsample, in a load sample, or in all GMP samples from the load takensubsequently during that run. Even without killing all of the microbes,the present invention can reduce a microbial bioburden to below GMPalert levels because of interference between the interaction between amicrobe and a chromatography matrix that also occurs during methods ofthe claimed invention. This interference may involve, but is not limitedto mechanisms such as reducing the affinity, binding, or any otherinteraction between the microbe and the chromatography matrix. Suchmechanisms are similar to the common strip step used on a chromatographycolumn that impurities such as host cell proteins and DNA from a column.Accordingly, this interference could be detected after use of a methodof the present invention, which does not kill all of the microbes, whenthe microbial bioburden has been reduced to levels consistent with therequirements of GMP production of a biologic pharmaceutical drug. Byusing one of the methods of the present invention that is not designedto kill all of the microbes on a column, the reagents are notnecessarily as harsh and therefore are more favorable for approval fromregulatory agencies, e.g., FDA or EMA, that have to approve the processand the product for market. Preferably, the same strip buffer componentsor at least the active agents that are used on a chromatography columnduring GMP production of a biologic pharmaceutical (acetic acid iscommon) can be used to reduce microbial bioburden when applied at ahigher concentration (for example, 10×, 15×, or 20×) and for a longercontract time (for example, 3×, 4×, or 5×). Further, it may be moreefficient for the same strip buffer to be used at a higher concentrationfor a longer time to reduce a microbial bioburden.

Any of the methods, aspects, and embodiments described herein can beused as part of a good manufacturing practice (GMP), or current goodmanufacturing practice (CGMP). Such practices must provide consistencyin manufacturing steps and quality of product so as to meet requirementsof regulatory bodies, such as the U.S. Food and Drug Administration. GMPand CGMP typically require a high degree of predictability andstandardization in manufacturing processes, particularly with ensuringpurity of the manufactured therapeutic biomolecules used in humanpatients. There are many failure points during a GMP or CGMP, in which aparameter is detected that requires stopping the process and/orscrapping the production batch. With the labor involved in growingcultures to produce biomolecules, great expense arises when a failureoccurs.

If the bioburden or microbial load gets too high in a chromatographycolumn, or matrix used in separation, various unpredictable and/orundesirable effects can arise. A failure point could be triggered so asto stop the process so that product contaminated with microbes isidentified and not further produced. To prevent failure points,detection of a bioburden of at least 5 CFU per 10 mL can trigger analert. A bioburden of at least 10 CFU per 10 mL can trigger action,which can include undertaking one or more of the methods or embodimentsdescribed herein, alone or in combination, to reduce the bioburden.

For example, microbes can be introduced into the product, rendering itunacceptable for therapeutic use. An excessive bioburden can alsodecrease column performance, which can interfere with purifying theproduct in a standardized and predictable way and possibly cause otherfailure points to be triggered. Therefore, it is desirable to use theherein described aspects and embodiments of reducing bioburdenpreemptively to ensure compliance with a GMP or CGMP, and to minimizefailure point triggering, and the associated troubleshooting anddowntime.

Any of the aspects or embodiments described herein may further comprisea step of applying a small molecule-containing or biomolecule-containing(e.g., monoclonal antibody-containing) preparation for purificationafter the contacting step. A method for reducing microbial load beforeapplying a small molecule-containing or biomolecule-containing (e.g.,monoclonal antibody-containing) preparation for purification cancomprise the steps of any of the methods of microbe bioburden reductiondescribed herein. Alternatively, a method for purifying a biomoleculecan comprise conducting the steps of any methods described herein andthen applying a preparation comprising the biomolecule to thechromatography matrix.

Any of the aspects or embodiments described herein may be performedafter the chromatography matrix is removed from storage but beforeapplication of a drug-containing, biomolecule-containing, or monoclonalantibody-containing preparation for purification. During long termstorage, a small amount of bacteria present in a chromatography matrixor column may grow and increase bioburden.

Any of the aspects or embodiments described herein may be used as partof an aseptic technique, or to support an aseptic technique. Theresulting reduction in bioburden on the chromatography matrix can besufficient for an aseptic technique, or can be used before or afterother steps in an aseptic technique. The described methods of reducingbioburden can reduce the odds that an aseptic technique failure pointwould be triggered and can be used in response to an impending failurepoint trigger.

In another aspect, a MabSelect™ Xtra chromatography matrix undergoesmicrobe bioburden reduction by contacting the matrix with a compositionconsisting essentially of about 0.5 M acetic acid, for at least 4 hours.In various embodiments, the contacting step is performed for 4 to 5hours, 4 to 10 hours, 4 to 25 hours, 4 to 200 hours, 4 to 375 hours, or4 to 400 hours. In another aspect, a MabSelect™ Xtra chromatographymatrix undergoes microbe bioburden reduction by contacting the matrixwith a composition consisting essentially of 0.1 M acetic acid and about20% ethanol, for at least 4 hours.

Various chromatography matrices may be used. The chromatography matrixmay comprise a proteinaceous ligand coupled to a support. Theproteinaceous ligand, in turn, may comprise one or more immunoglobulinbinding domains. Other useful chromatography matrices include, withoutlimitation, various ion exchange chromatography matrices, hydrophobicinteraction chromatography (HIC) matrices, mixed mode chromatographymatrices, and size exclusion chromatography matrices.

The proteinaceous ligand of the chromatography matrix may be Protein Aor a fragment or a derivative thereof. Exemplary proteinaceous ligandsinclude Staphylococcus Protein A, Peptostreptococcus Protein L,Streptococcus Protein G, Streptococcus Protein A, and fragments andderivatives of any of Staphylococcus protein A, PeptostreptococcusProtein L, Streptococcus Protein G, Streptococcus Protein A.

Staphylococcus Protein A may be found on the cell wall of the bacteriaStaphylococcus aureus. Protein A may bind antibodies in the Fc region,between the CH2 and CH3 domains. Protein A may be cultured inStaphylococcus aureus or produced recombinantly in other bacteria, forexample, E. coli or Brevibacillus. Fragments or derivatives ofStaphylococcus Protein A may also bind to antibodies in the Fc region,between the CH2 and CH3 domains.

Peptostreptococcus Protein L may be found on the surface ofPeptostreptococcus magnus and can bind to antibodies via an interactionwith the antibody light chain. Unlike Protein A, Protein L may bind tosingle chain variable fragments (scFv) and Fab fragments. Fragments orderivatives of Protein L may also bind to the light chain of antibodies,single chain variable fragments (scFv) and Fab fragments.

Streptococcus Protein G may be found on the cell wall of group GStreptococcal strains. Protein G may bind antibodies in the Fab and Fcregions. Protein G may be produced recombinantly in other bacteria, forexample, E. coli. Fragments or derivatives of Protein G may also bind toantibodies in the Fab and Fc regions.

The chromatography matrix may be a resin that is part of a column. Onesuitable resin is MabSelect SuRe™ from GE Healthcare. An exemplarycolumn suitable for small scale purifications is packed with Mab SelectSuRe™, is about 1.0 cm in diameter, and about 20 cm long. A largercolumn, such as 1.4 m×20 cm, can be used for manufacturing scalepurifications.

More generally, the methods of the present invention can be used forchromatography columns of various sizes, including laboratory scale,large process scale, and very large process scale. In some embodiments,the chromatography column may have an inner diameter between 0.5 cm to1.5 cm and a bed height of between 15 to 30 cm (e.g., 20 cm). The innerdiameter may be between 0.7 to 1.2 cm, alternatively 0.9 to 1.4 cm, 1.2to 1.5 cm, 1.0 to 1.2 cm, or about 1 cm. In some embodiments, thechromatography column may have an inner diameter of between 40 cm to 1.6meters (e.g., 60 cm, 80 cm, 1.0 meter, 1.2 meters, or 1.4 meters). Thechromatography column may have a bed height of between 15 to 30 cm(e.g., 20 cm).

Exemplary chromatography matrices include MabSelect™, MabSelect™ Xtra,MabSelect™ SuRe, MabSelect™ SuRe pcc, MabSelect™ SuRe LX, nProtein ASepharose 4 Fast Flow, Protein A Sepharose 4 Fast Flow, Protein A MagSepharose, Protein A Sepharose CL-4B, rmp Protein A Sepharose Fast Flow,rProtein A Sepharose 4 Fast Flow, Capto™ L, ProSep™-A, ProSep UltraPlus, AbSolute™, CaptivA™ PriMab™, Protein A Diamond, Eshmuno™ A,Toyopearl™ AF-rProtein A, Amsphere™ Protein A, KanCapA™, Protein G MagSepharose Xtra, and Protein G Sepharose 4 Fast flow.

MabSelect™, MabSelect™ Xtra, MabSelect™ SuRe, and MabSelect™ SuRe LXhave a recombinant protein A ligand, produced in E. coli, that isattached to a highly cross-linked agarose matrix.

Microbe bioburden reduction can restore performance of a chromatographymatrix so that it can be used for additional purification rather thanbeing replaced. Thus, in any of the methods described herein, microbebioburden reduction can be conducted after the chromatography matrix hasbeen used. In such scenarios, bacteria and other microorganisms that maybe introduced into the chromatography matrix from cell culture brothscomprising a monoclonal antibody of interest, can be removed.Substantial expense can be saved when using chromatography matricescomprised of proteinaceous ligands, such as Protein A.

Furthermore, using acetic acid-containing solutions instead of commonlyused sodium hydroxide-containing solutions can result in lessdenaturation of the protein ligand and less damage to the chromatographymatrix. Denaturation of the protein ligand and/or damage to thechromatography matrix can be measured indirectly by assaying variousperformance characteristics of the chromatography matrix, e.g., byassaying the purity and abundance of the product that elutes from thematrix and by assaying residual elution of a component of the matrix(e.g., Protein A). For example, if the proteinaceous ligand is ProteinA, the purity and abundance of monoclonal antibodies would be assayed.Exemplary assays include size exclusion chromatography (e.g., SE-HPLCand SE-UPLC), capillary electrophoresis (e.g., CE-SDS), capillaryisoelectric focusing (iCIEF) that can optionally include whole columnimaging. Also, residual Protein A from the column can be measured (e.g.,by an assay comprising ELISA).

Microbe bioburden reduction undertaken according to the methodsdescribed herein can be a cost-effective way to maintain performance ofa column comprising Protein A or other proteinaceous ligands, byremoving bacteria without damaging the Protein A. For example, exposureof a Protein A-containing matrix to an acetic acid-comprising solution(e.g., 0.5 M acetic acid) for 375 or 400 hours does not lead to anystatistically significant loss of performance of a column comprisingProtein A. See, e.g., Example 6, Table 10 and FIGS. 5-19. For example,there is no statistically significant loss of purity of elutedmonoclonal antibody by size exclusion chromatography, capillaryelectrophoresis under reducing or non-reducing conditions, or bycapillary isoelectric focusing.

Microbe bioburden reduction according to the methods described hereincan remove nearly all of the bacteria without killing all of thebacteria. Without wishing to be bound by theory, acetic acid caninterfere with the affinity between bacteria and the proteinaceousligand, e.g., protein A. A microbe-contaminated matrix or column canhave microbes reduced according to methods described herein bydisrupting interactions between microbial organisms and the resin. Thebacteria would tend to remain in the acetic acid-containing solution.Removal of the bacteria may be further enhanced by repeating thecontacting steps with acetic acid or undertaking additional flushing ofthe chromatography matrix with acetic acid-containing solutions.

In some embodiments, the method removes spore-forming bacteria from thechromatography matrix. Spore-forming bacteria have the ability to switchto endospore form. Endospore form is a stripped-down, dormant form towhich the bacterium can reduce itself. Endospore formation is usuallytriggered by a lack of nutrients or harsh conditions, such as acidic orbasic environment. Endospores enable bacteria to lie dormant forextended periods even in unfavorable conditions. When the environmentbecomes more favorable, the endospore can reactivate to the vegetativestate. Examples of bacteria that can form endospores include Bacillusand Clostridium. These spore-forming bacteria are thought to be able toform endospore under normal operating conditions in chromatographypurification processes due to the existence of unfavorable conditionsfor these spore-forming bacteria in the manufacturing process. There aremany methods that can be used to detect spore-forming bacteria and thoseinclude but not limited to microscopic bacterial staining method,IR/FTIR spectroscopy method, sterility tests, and bacterialidentification tests (e.g., biochemical reactions, 16S rRNA sequencedetermination, or taxa-specific sequence determinations).

In some embodiments, the method removes Gram positive bacteria from thechromatography matrix. In some embodiments, the method can remove Gramnegative bacteria from the chromatography matrix. In some embodiments,the method removes spore-forming bacteria, Gram positive bacteria andGram negative bacteria from the chromatography matrix. The contactingstep may result in a reduction in the amount of one or more of sporeforming bacteria, gram positive bacteria, and gram negative bacteria, inthe chromatography matrix, to below the limit of detection of an assayselected from the group consisting of (1) a biofiltration assay, (2)microscopic bacterial staining, (3) IR/FTIR spectroscopy method, (4) asterility test, and (5) a bacterial identification test (e.g., abiochemical reaction, 16S rRNA sequence determination, or ataxa-specific sequence determination). Biofiltration assays aredescribed in the U.S. Pharmacopeia Chapter <71>, titled “SterilityTests.” In biofiltration assays, elutions from the column are passedthrough a filter that selectively binds bacteria. The filter is thenplated on agar with appropriate media to grow bacteria, incubated, andthe bacteria counted. Dilutions of the column elution can be performedas needed.

Microscopic bacteria staining is described in the U.S. PharmacopeiaChapter <61>, titled “Microbial examination of nonsterile products:microbial enumeration tests”. IR/FTIR spectroscopy methods are describedin BRUKER Application Note AN #405 Current Research, Technology andEducation Topics in Applied Microbiology Biotechnology A. Mendez-Vilas(Ed.) Microbiological tests are described in Reynolds, J. et al.,“Differential staining of bacteria: endospore stain” Curr. Proc.Microbiol. 2009, Appendix 3: Appendix 3J.

In some embodiments, the concentration of acetic acid in the compositionis from about 0.1 M to about 1.0 M. In some embodiments, theconcentration of acetic acid in the composition is from about 0.2 M toabout 0.8 M. In some embodiments, the concentration of acetic acid inthe composition is from about 0.4 M to about 0.7 M. In some embodiments,the concentration of acetic acid in the composition is about 0.5 M. Insome embodiments, the concentration of acetic acid in the composition isfrom about 0.1 M to about 0.5 M. In some embodiments, the concentrationof acetic acid in the composition is about 0.1 M.

In some embodiments, the concentration of urea in the composition isfrom about 4 M to about 12 M. In some embodiments, the concentration ofurea in the composition is from about 6 M to about 10 M. In someembodiments, the concentration of urea in the composition is from about6 M to about 8 M. In some embodiments, the concentration of urea in thecomposition is about 8 M.

In some embodiments, the concentration of guanidine hydrochloride in thecomposition is from about 3 M to about 10 M. In some embodiments, theconcentration of guanidine hydrochloride in the composition is fromabout 4 M to about 8 M. In some embodiments, the concentration ofguanidine hydrochloride in the composition is from about 5 M to about 7M. In some embodiments, the concentration of guanidine hydrochloride inthe composition is about 6 M.

In some embodiments, the concentration of guanidine hydrochloride in thecomposition is from about 3 M to about 10 M. In some embodiments, theconcentration of guanidine hydrochloride in the composition is fromabout 4 M to about 8 M. In some embodiments, the concentration ofguanidine hydrochloride in the composition is from about 5 M to about 7M. In some embodiments, the concentration of guanidine hydrochloride inthe composition is about 6 M.

In some embodiments, the pH of the solution is at least 2.0. The pH maybe from 2.0 to 7.0. The pH may be from 2.5 to 6.5, from 3.0 to 6.0, from4.0 to 7.0, from 2.0 to 5.0, from 3.5 to 5.5, from 3.0 to 4.0, or about4.0. The pH may be about any of the following: 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, or 7.0.

In some embodiments, the composition comprises ethanol. The compositionmay comprise 1-40% ethanol, 5-35% ethanol, 10-25% ethanol, 15-30%ethanol, 18-24% ethanol, about 20% ethanol, or 20% ethanol. In someembodiments, the composition comprises about 0.1M acetic acid and about20% ethanol. In some embodiments, the composition consists essentiallyof about 0.1M acetic acid and about 20% ethanol. There are advantages tousing ethanol, while and minimizing the amount of benzyl alcohol used,or avoiding benzyl alcohol due to toxicity in humans, or adverse effectsin humans, that may arise from presence of benzyl alcohol.

In some embodiments, the composition further comprises an acetate salt.The acetate salt may serve as a buffer for compositions comprisingacetic acid. For example, the composition may comprise sodium acetate inaddition to acetic acid such that the composition is a buffer. Thecomposition may comprise from 0.1 M sodium acetate to 1.0 M sodiumacetate, 0.2 to 0.8 M sodium acetate, 0.4 to 0.7 M sodium acetate, about0.5 M sodium acetate, or 0.5 M sodium acetate. Buffering the acetic acidwith sodium acetate or another acetate salt may be effective to maintainthe pH of the solution in the chromatography matrix. The pH may be atleast 2.0, from about 2 to 3, from 2.0 to 3.0, from 2.0 to 7.0, from 2.5to 6.5, from 3.0 to 6.0, from 4.0 to 7.0, from 2.0 to 5.0, from 3.5 to5.5, from 3.0 to 4.0, or about 4.0. The pH may be about any of thefollowing: 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

In some embodiments in which the composition comprises acetic acid, thecontacting step is performed for at least one hour, alternatively for 1to 4 hours, alternatively for at least 2 hours. In some embodiments, thecontacting step is performed for 2 to 4 hours. In some embodiments, thecontacting step is performed for at least 4 hours. In variousembodiments, the contacting step is performed for 90 minutes to 6 hours,2 hours to 5 hours, 4 hours to 5 hours, 4 hours to 6 hours, 90 minutesto 3 hours, 5 hours to 6 hours, 4 hours to 10 hours, 4 hours to 25hours, 4 hours to 200 hours, 4 hours to 375 hours, or 4 hours to 400hours.

In some embodiments in which the composition comprises urea, thecontacting step is performed for at least 30 minutes, alternatively atleast one hour, alternatively for at least two hours. In someembodiments, the contacting step is performed for one to two hours. Insome embodiments, the contacting step is performed for at least twohours. In various embodiments, the contacting step is performed for 30minutes to 4 hours, 1 hour to 3 hours, or 90 minutes to 2 hours.

In some embodiments in which the composition comprises guanidinehydrochloride, the contacting step is performed for at least 30 minutes,alternatively at least one hour, alternatively for at least two hours.In some embodiments, the contacting step is performed for one to twohours. In some embodiments, the contacting step is performed for atleast two hours. In various embodiments, the contacting step isperformed for 30 minutes to 4 hours, 1 hour to 3 hours, or 90 minutes to2 hours.

In some embodiments in which the composition comprises urea or guanidinehydrochloride, the contacting step is performed for at least 30 minutes,alternatively for at least about 1 hour, alternatively for 1 to 4 hours,alternatively for at least 2 hours.

In some embodiments, the contacting step can be repeated. For example,the contacting step may be performed for about one hour and thenrepeated multiple times. In some embodiments, the contacting step isrepeated 2, 3, 4, 5, or 6 times. By repeating the microbe bioburdenreduction, the column may be exposed to additional acetic acid, whichcan result in additional disruption of bacteria and microorganisms fromthe chromatography matrix. Repeating the contacting step can lead tomore flushing, or removal, of bacteria and microorganisms from thechromatography matrix, e.g., a Protein A ligand, and the column. Themicrobe bioburden reduction process may reduce bioburden more whenconducted multiple times in succession.

The effectiveness of any of the microbe bioburden reduction methodsdescribed herein can be monitored by using any number of bioburdenassays. One such assay is a filtration assay where a volume of eluate ispassed through a filter membrane that traps the bacteria present in theeluate. The bacteria titer can be determined by placing the filtermembrane on agar plates such that the bacteria on the filter membraneform colonies on the plates. The agar plates may contain trypticase soyagar (TSA). Culturing may occur for 3 to 7 days at temperatures between25° C. and 37° C. The number of colonies is then counted. If there aretoo many colonies formed, dilutions of the eluate can be undertaken.

In some embodiments, there is a reduction in the amount of spore formingbacteria by at least 1.5 log₁₀. For example, when a chromatographymatrix is contaminated with Bacillus pseudofirmus, contacting suchmatrix with an 8 M urea solution, 8 M urea/20% ethanol solution, a 6 Mguanidine hydrochloride solution, or a 6 M guanidine hydrochloride/20%ethanol solution, for at least one hour can reduce the number ofBacillus pseudofirmus by at least 1.5 log₁₀.

In some embodiments, the binding capacity of the chromatography matrixis preserved over 10 or more cycles when the method for microbialbioburden reduction is undertaken. In other embodiments, the bindingcapacity is preserved over 50 or more cycles. In some other embodiments,the binding capacity is preserved over 100 or more cycles. In someembodiments, the binding capacity is preserved over 200 or more cycles.

In some embodiments, there is no substantial degradation of thechromatography matrix during the contacting step or over multiplecontacting steps wherein the exposure to the composition is for at least5 hours, at least 10 hours, at least 25 hours, at least 200 hours, atleast 375 hours, or at least 400 hours. In some embodiments, there is nomeasurable degradation of the chromatography matrix during thecontacting step or over multiple contacting steps wherein the exposureto the composition is for at least 5 hours, at least 10 hours, at least25 hours, at least 200 hours, at least 375 hours, or at least 400 hours.In some embodiments, the degradation of the chromatography matrix ismeasured by the protein quality of a protein that binds to the matrix(e.g., a monoclonal antibody that binds to a Protein A matrix).

In some embodiments, the proteinaceous ligand is not measurablydenatured. Denaturation can be determined using functional assays suchas, e.g., measuring column performance, product yield and/or quality,leachable proteinaceous ligand from the matrix, denaturation of theproteinaceous ligand, etc.

In some embodiments, there is no measurable leaching of theproteinaceous ligand, or Protein A during the contacting step or overmultiple contacting steps. In some embodiments, there is nostatistically significant measurable leaching of the proteinaceousligand, or Protein A after exposure of the chromatography matrix to thecomposition (e.g., 0.5 M acetic acid) for 5 hours, 10 hours, 25 hours,200 hours, 375 hours or 400 hours. Measurement of leaching of Protein Ais one such exemplary assay. Denaturation of Protein A can change itsconfirmation such that it no longer interacts with beads or other solidphase support in the chromatography matrix. The denatured Protein A thenwould leach off of the beads or solid support and enter the liquidphase. Detection of the proteinaceous ligand, or Protein A, in theeluate or liquid phase of the affinity column can thus indicatemeasurable denaturation. Denaturation of other proteinaceous ligands,besides Protein A, may also lead to their leaching from thechromatography matrix. In some embodiments, the measurement of leachingof Protein A and/or other proteinaceous ligands comprises ELISA. Anexemplary assay is described in Example 6 and FIGS. 8, 13 and 18.

EXAMPLES

The following examples describe the various aspects and embodimentsdescribed above. However, the use of these and other examples anywherein the specification is illustrative only and in no way limits the scopeand meaning of any of the disclosure or of any exemplified term.Likewise, any claimed subject matter is not limited to any particularpreferred embodiments described here. Indeed, many modifications andvariations may be apparent to those skilled in the art upon reading thisspecification, and such variations can be made without departing inspirit or in scope from the aspects and embodiments disclosed herein.Any claimed subject matter is therefore to be limited only by the termsof the appended claims along with the full scope of equivalents to whichthose claims are entitled.

Example 1 Acetic Acid Solution Reduces Bioburden in a Protein A Column

Reduction of microbes in three packed 1 cm MabSelect™ Xtra columns wasassessed by measuring the bioburden after the column is spiked with aparticular bacterium and then again after reducing microbes with a 0.5 Macetic acid solution.

First, the column was flushed with two column volumes of water forinjection (WFI). A 20 mL sample was collected and served as a negativecontrol with respect to the amount of bacteria in the column.

In each of the three MabSelect™ Xtra columns, a representativemicroorganism was added to the column by adding the microorganism to WFIto form spiked \WI, wherein the microorganism is present in a titer ofapproximately 10⁵ cfu/mL. One column was spiked with spore-formingbacteria, particularly Bacillus psuedofirmus. One column was spiked withGram positive bacteria, particularly Microbacterium spp. The thirdcolumn was spiked with Gram negative bacteria, particularlyStenotrophomonas maltophilia.

Each spiked WFI was then loaded onto each column. The columns were thenflushed with 14 column volumes of WFI at 229 cm/hour, which werecollected. Each column was held for one hour and then flushed again withthe spiked WFI. A 20 mL sample was collected from each column as apositive control, with the amount of each of the Gram negative bacteria,Gram positive bacteria and spore-forming bacteria in the samplesubsequently assayed.

Two column volumes of a microbe reducing solution of 0.5 M acetic acidwere then applied to each column at 229 cm/hour. Each column was heldfor one hour and then flushed with two column volumes of WFI at 229cm/hour. For Microbacterium spp. and Stenotrophomonas maltophilia, 1.5column volumes of WFI were flushed through the column, then 1.5 columnvolumes of effluent were collected. For Bacillus psuedofirmus, 2 columnvolumes of an equilibration buffer (10 mM Sodium Phosphate, 500 mMSodium Chloride, pH7.2) were flushed through the column, then 1.5 columnvolumes of effluent are collected.

The above experiment was repeated, with the columns held for four hoursinstead of one hour after the 0.5 M acetic acid microbe reducingsolution was applied to the column.

A filtration-based bioburden assay was performed. Agar plates areprepared that have TSA media.

All of the above samples from the chromatography column were placed intoa conical tube, inverted 10 times, and then passed through a filtermanifold connected to sterile single use filter funnels having a 0.45micron filter membrane, or a Milliflex® plus pump with a Milliflex®filter funnel unit having a 0.45 micron filter membrane. Steriletechnique was used in handling the filter manifold or filter funnel unitso as not to introduce additional bacteria not found in thechromatography sample. Each membrane was then placed on top of the agarplate prepared with TSA media. The plates were incubated for 5-7 days at30-35° C. The number of colony forming units was then counted andrecorded.

Negative control plates are prepared by passing 100 mL of sterile PBSinto a separate filter manifold or filter funnel unit with a 0.45 micronfilter membrane. Each membrane is then placed on top of the agar plateprepared with TSA media. The plates were incubated for 5-7 days at30-35° C. The number of colony forming units was then counted andexpressed in a log₁₀ format.

The tables below show the colony forming units pre-reduction andpost-reduction for each of the three bacteria types.

TABLE 1 Bacillus Bacillus pseudofirmus pseudofirmus MicrobeSaturation/Pre- Post-Microbe Bioburden Microbe Bioburden MicrobeBioburden Reduction Bioburden Reduction Reduction Solution DurationReduction (log₁₀) (log₁₀) 0.5M acetic acid 1 hour 1.5 1.1 0.5M aceticacid 4 hour 3.4 0

TABLE 2 Microbacterium spp. Microbacterium Saturation/ spp. MicrobePre-Microbe Post-Microbe Bioburden Bioburden Bioburden Microbe BioburdenReduction Reduction Reduction Reduction Solution Duration (log₁₀)(log₁₀) 0.5M acetic acid 1 hour 5.1 3.7 0.5M acetic acid 4 hours 5.6 0

TABLE 3 Stenotrophomonas maltophila Saturation/ Stenotrophomonas MicrobeMicrobe Pre-Microbe maltophila Bioburden Bioburden BioburdenPost-Microbe Reduction Reduction Reduction Bioburden Solution Duration(log₁₀) Reduction (log₁₀) 0.5M acetic acid 1 hour 5.5 0 0.5M acetic acid4 hours 6.2 0

For the Bacillus pseudofirmus, a 0.4 log₁₀ reduction was observed whenthe 0.5 M acetic acid microbe bioburden reduction solution was held inthe column for one hour, A 3.4 log₁₀ reduction was observed when the 0.5M acetic acid microbe bioburden reduction solution was held in thecolumn for four hours.

For the Microbacterium spp. bacteria, a 1.4 log₁₀ reduction was observedwhen the 0.5 M acetic acid microbe bioburden reduction solution was heldin the column for one hour, and a 5.6 log₁₀ redaction was observed whenheld for four hours.

For the Stenotrophomonas maltophila bacteria, a 5.5 log₁₀ reduction wasobserved when the 0.5 M acetic acid microbe bioburden reduction solutionwas held in the column for one hour, and a 6.2 log₁₀ reduction wasobserved when held for four hours.

0.5 M acetic acid is effective to remove a wide range of microorganisms,including spore-forming bacteria, when held in a protein A column forfour hours.

Example 2 Acetic Acid/Ethanol Solution Reduces Bioburden in a Protein AColumn

The steps in Example 1 above were undertaken, except that instead of 0.5M acetic acid, a solution of 0.1 M acetic acid and 20% ethanol was used.As with Example 1, the 0.1 M acetic acid and 20% ethanol solution washeld for one hour in one set of experiments and for four hours inanother set of experiments. The results below also show a substantialdecrease in the amount of bacteria after the microbe bioburden reductionsolution was held in the column for either one or four hours.

TABLE 4 Bacillus Bacillus Microbe pseudofirmus pseudofirmus BioburdenSaturation/Pre- Post-Microbe Microbe Bioburden Reduction MicrobeBioburden Bioburden Reduction Solution Duration Reduction (log₁₀)Reduction (log₁₀) 0.1M acetic acid 1 hour 2.2 1.8 and 20% ethanol 0.1Macetic acid 4 hours 2.5 0 and 20% ethanol

TABLE 5 Microbacterium spp. Microbacterium Microbe Saturation/ spp.Bioburden Pre-Microbe Post-Microbe Microbe Bioburden Reduction BioburdenBioburden Reduction Solution Duration Reduction (log₁₀) Reduction(log₁₀) 0.1M acetic acid 1 hour 5.1 0.6 and 20% ethanol 0.1M acetic acid4 hours 5.1 0 and 20% ethanol

TABLE 6 Stenotrophomonas maltophila Stenotrophomonas Microbe MicrobeSaturation/ maltophila Bioburden Bioburden Pre-Microbe Post-MicrobeReduction Reduction Bioburden Bioburden Solution Duration Reduction(log₁₀) Reduction (log₁₀) 0.1M acetic acid 1 hour 5.3 0 and 20% ethanol0.1M acetic acid 4 hours 5.1 0 and 20% ethanol

For the Bacillus pseudofirmus, a 0.4 log₁₀ reduction was observed whenthe 0.1 M acetic acid and 20% ethanol microbe bioburden reductionsolution was held in the column for one hour. A 2.5 log₁₀ reduction wasobserved when the 0.1 M acetic acid and 20% ethanol microbe bioburdenreduction solution was held in the column for four hours.

For the Microbacterium spp. bacteria, a 4.5 log₁₀ reduction was observedwhen the 0.1 M acetic acid and 20% ethanol microbe bioburden reductionsolution was held in the column for one hour, and a 5.1 log₁₀ reductionwas observed when held for four hours.

For the Stenotrophomonas maltophila bacteria, a 5.3 log₁₀ reduction wasobserved when the 0.1 M acetic acid and 20% ethanol microbe bioburdenreduction solution was held in the column for one hour, and a 5.1 log₁₀reduction was observed when held for four hours.

Example 3 A Urea Solution Reduces Bioburden in a Protein A Column

The steps in Example 1 above were undertaken, except that instead of 0.5M acetic acid, a solution of 8 M urea and a solution of 8 M urea/20%ethanol were used and only a one hour hold was measured. The results inTable 7 below show a substantial decrease in bacteria after the microbebioburden reduction solution was held in the column for one hour. Thereduction in spore-forming B. psuedofirmus was extensive and unexpected,particularly after only one hour of treatment.

TABLE 7 Microbial Bioburden Log₁₀ of Reduction Reduction Hold BacillusMicrobacterium Stenotrophomonas Solutions time pseudofirmus speciesmaltophilia 8M Urea 1 hr 1.9 5.8 5.7 8M Urea/ 1 hr 1.6 5.7 6.7 20%Ethanol

Example 4 A Guanidine Hydrochloride Solution Reduces Bioburden in aProtein A Column

The steps in Example 1 above were undertaken, except that instead of 0.5M acetic acid, a solution of 6 M guanidine hydrochloride and a solutionof 6 M guanidine hydrochloride/20% ethanol were used and only a one hourhold was measured. The results in Table 8 below show a substantialdecrease in bacteria after the microbe bioburden reduction solution washeld in the column for one hour. The reduction in spore-forming B.psuedofirmus was extensive and unexpected, particularly after only onehour of treatment.

TABLE 8 Microbial Bioburden Log₁₀ of Reduction Reduction Hold BacillusMicrobacterium Stenotrophomonas Solutions time pseudofirmus speciesmaltophilia 6M 1 hr 1.6 5.4 2.4 Guanidine Hydro- chloride 6M 1 hr 2.04.7 4.7 Guanidine Hydro- chloride/ 20% Ethanol

Example 5 Tests of Microbial Bioburden Reduction Agents in Solution

A solution spike study was performed using 0.5 M acetic acid, where theextent of killing of Bacillus psuedofirmus and Microbacterium specieswas measured in solution, without chromatography matrix present. Thedata are illustrated in FIG. 1. There was little killing of Bacilluspsuedofirmus observed after one hour, while there was some killing ofMicrobacterium species. These data illustrate that killing is not solelyresponsible for the microbial bioburden reduction of these bacteria, ona chromatography matrix, with 0.5 M acetic acid. Disruption of aninteraction between the chromatography matrix and Bacillus psuedofirmusand Microbacterium species leads to increased reduction of bioburdenthan what would be expected from killing.

Additional solution spike studies were performed using other agents,where the extent of killing of Bacillus psuedofirmus, Microbacteriumspecies, and Stenotrophomonas maltophilia were measured in solution. Thefollowing agents were added: (a) water for injection (WFI), (b) 8 Murea, (c) 8 M urea and 20% ethanol, (d) 6 M guanidine hydrochloride, (e)6 M guanidine hydrochloride with 20% ethanol. A spike confirmationmeasurement in PBS was taken, as well as measurements at the 0 minute,30 minute, and 60 minute time points. The data are shown in FIGS. 2-4,where the black bar is for WFI, the horizontally striped bar is for 8 Murea, the white bar is for 8 M urea and 20% ethanol, the diagonallystriped bar is for 6 M guanidine hydrochloride and the cross-hatched baris for 6 M guanidine hydrochloride and 20% ethanol.

For Bacillus pseudofirmus, the bioburden reduction is achieved by acombination of killing and disrupting of interactions between microbesand chromatography resin with at least the following solutions: 0.5 Macetic acid, 8 M urea and 8 M urea/20% ethanol, 6 M guanidinehydrochloride and 6 M guanidine hydrochloride/20% ethanol.

For Microbacterium species, bioburden reduction may occur by acombination of killing and disrupting interactions for 8 M urea and 0.5M acetic acid, which are solutions that are not able to kill 100% ofMicrobacterium species. However, killing may be largely responsible forbioburden reduction with 8 M urea/20% ethanol, 6 M guanidinehydrochloride and 6 M guanidine hydrochloride/20% ethanol, which wereobserved to kill 100% of the Microbacterium in solution. Similarly forStenotrophomonas maltophilia, 8 M urea, 8 M urea/20% ethanol were ableto kill 100% Stenotrophomonas maltophilia in solution.

Examples 1-5 in summary show that 0.5 M acetic acid with a 4-hour hold,8 M urea with a 1-hour hold and 8 M urea/20% ethanol with a 1-hour hold,6 M guanidine hydrochloride with 1-hour hold and 6 M guanidinehydrochloride/20% ethanol with 1-hour hold were discovered to be aneffective microbial bioburden reduction method for packed MabSelect™Xtra column in manufacturing. As described in Examples 1-5, these agentsare effective through the combination of killing the microbes anddisrupting the interaction between microbial organisms andchromatography resin, or 100% to killing microbial organisms.

Additionally, MabSelect™ Xtra resin exposure to 0.5 M acetic acidresulted in minimal impact on Protein A resin.

Example 6 Affinity Columns Maintain Performance after Prolonged Exposureto Acetic Acid

The performance characteristics of two different chromatography affinityresins, MabSelect™ Xtra and MabSelect™ SuRe, were assessed after theresins were soaked in 0.5 M acetic acid for various lengths of time.Specifically, one fraction of MabSelect™ Xtra (used to capture mAb A)was soaked in 0.5 M acetic acid for 375 hours. As a negative control,another fraction of MabSelect™ Xtra was not soaked in 0.5 M acetic acid.Five different fractions of MabSelect™ SuRe (used to capture mAb B) weresoaked in 0.5 M acetic acid for each of 5 hours, 10 hours, 25 hours, 200hours, or 400 hours. As a control, another fraction of MabSelect™ SuRewas not soaked in 0.5 M acetic acid.

Five different experiments were then conducted on each of the abovefractions above to assess performance. Size exclusion chromatography(SE-HPLC and SE-UPLC) was performed to assess mAb purity afterpurification on each of the above chromatography affinity resins.

TABLE 9 Flow Rate Temperature Type of Column Mobile Phase Buffer (cm/hr)(° C.) MabSelect Xtra 10 mM Sodium Phosphate, 229 20-25 500 mM SodiumChloride MabSelect SuRe 10 mM Sodium Phosphate, 231 20-25 500 mM SodiumChloride

Two different capillary electrophoresis experiments were performed.CE-SDS was conducted for mAb A and PICO Microchip CE-Electrophoresis(PICO MCE-SDS) was conducted for mAb B. Capillary electrophoresis wasconducted in SDS-containing gel-filled capillaries (CE-SDS) to measurethe molecular weight distribution and relative abundance of light andheavy chain from monoclonal antibodies. These proteins were separatedbased on their size and electrophoretic mobility. The relative abundanceof total light chain and heavy chain was conducted under reducing andnon-reducing conditions. CE-SDS was performed using the IgG PurityAnalysis Kit (Beckman Coulter, A10663) with a Bare Fused SilicaCapillary (capillary length 57 cm, effective length 50 cm). PICO MCE-SDSwas performed using Protein Express LabChip, LabChip® GXII, or LabChip®GXII Touch HT (Perkin Elmer, 760499 or 760528). Internal standards wereused to calibrate the relative migration time.

To assay for effect of acetic acid on the Protein-A containing matrix, aresidual protein A analysis was performed on eluates from the MabSelect™Xtra and MabSelect™ SuRe columns by high throughput ELISA andquantified.

Capillary isoelectric focusing with whole-column imaging (iCIEF) wasperformed to quantify the amount of complementarity determining region 2(CDR2) in a monoclonal antibody sample. Relative abundance of the CDR2was calculated in each electropherogram by integrating the area undereach of the sample-derived isoelectric point (pi) distribution peaksobserved and calculating the percentage attributable to CDR2. Thereported iCIEF Region 2 is the principal peak of neutral species andcorresponds to the largest protein peak in the internal referencestandard.

The results of each of the above analyses are shown in the followingTable 10.

TABLE 10 Hours of CE-SDS Exposure SE- Reduced CE-SDS to 0.5M UPLC TotalLC + Non- iCIEF Acetic Purity HC Purity Reduced Protein Region 2 ResinAcid (%) (%) Purity (%) A (ppm) (%) MabSelect 0 95.00 92.77 92.41 6.1540.40 Xtra (used 95.00 92.74 92.37 5.28 40.00 for capture 95.00 92.8192.52 5.45 41.10 of mAb A) 375 95.00 92.75 92.65 6.11 41.40 95.00 92.4492.18 6.93 40.60 95.00 92.60 91.96 6.02 39.10 MabSelect 0 94.27 95.5089.00 3.50 45.20 SuRe 94.62 94.70 90.10 4.50 50.20 (used for 93.81 94.1090.20 2.80 49.80 capture of 93.99 96.00 89.00 5.30 44.60 mAb B) 93.6195.70 89.50 4.70 45.00 93.78 95.50 89.10 4.90 45.70 94.76 95.80 89.404.30 45.00 94.56 95.80 89.40 5.40 45.20 95.13 95.70 89.10 3.70 45.2093.54 95.00 89.60 4.10 45.50 93.40 94.70 89.60 2.70 45.50 91.93 96.5089.60 1.90 47.80 93.98 95.20 89.90 4.50 48.60 93.58 95.90 89.20 3.2048.90 93.16 95.20 89.30 3.30 48.60 5 94.76 95.30 90.30 3.70 49.60 91.0195.00 90.10 4.10 50.30 94.54 94.80 90.30 2.90 49.50 10 94.54 94.80 89.703.70 49.50 94.21 94.40 89.80 3.50 49.50 94.52 94.60 90.50 4.50 49.20 2593.22 94.80 88.60 3.40 45.60 94.24 95.70 88.90 4.70 45.20 94.41 95.6089.60 6.10 45.60 200 93.82 95.60 89.90 3.20 47.80 93.70 96.80 89.30 2.2046.90 94.40 95.30 89.90 2.20 46.50 400 94.84 95.90 89.50 3.80 47.4094.76 94.50 90.10 27.70 48.40 94.81 93.90 89.70 3.60 48.10

The data from the above table are shown in each of FIGS. 5 through 9.Visual inspection of these figures shows no negative correlation betweenthe performance characteristic and the length of time exposed to 0.5 Macetic acid.

Analysis of variance (ANOVA) for the product quality data was performedto assess for statistically significant differences between the resinsbefore and after prolonged exposure to 0.5 M acetic acid using threechromatography runs per resin condition. FIGS. 10 through 14 showprotein quality of the resultant mAb A pool after Mab Select Xtrapurifications.

FIGS. 15 through 19 show protein quality of the resultant mAb B poolafter MabSelect SuRe purifications. ANOVA analysis of protein qualityshows no statistical significant (p<0.05) besides in the SE-UPLC percentpurity of the mAb B MabSelect SuRe pool. However, the post-acid poolpurity is higher than the pre-acid pool purity. Therefore, there is nonegative effect on the mAb B pool after purification with MabSelect SuRethat has prolonged exposure to 0.5 M acetic acid.

The claimed subject matter is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theclaimed subject matter in addition to those described herein will becomeapparent to those skilled in the art from the foregoing description.Such modifications are intended to fall within the scope of the appendedclaims.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference intheir entirety as if physically present in this specification.

The invention claimed is:
 1. A method for microbial bioburden reductionof a chromatography matrix, comprising: contacting a chromatographymatrix with a composition, wherein the composition consists of: (a) 0.4M to 0.7 M acetic acid; (b) 0.4 M to 0.7 M acetic acid and benzylalcohol; (c) 0.1 M to 0.5 M acetic acid and 1% benzyl alcohol; or (d)0.5 M to 1.0 M acetic acid and 1% to 2% benzyl alcohol, wherein thecontacting step is performed for 1 hour to 4 hours, and the method doesnot comprise a step of contacting the chromatography matrix with asodium hydroxide solution.
 2. The method of claim 1, wherein thecontacting step is performed for 4 hours.
 3. The method of claim 1,wherein the composition (a) consists of 0.5 M acetic acid, and whereinthe contacting step is performed for 4 hours.
 4. A method for microbialbioburden reduction of a chromatography matrix, comprising: contacting achromatography matrix with a composition, wherein the compositionconsists of: (a) 0.4 M to 0.7 M acetic acid; (b) 0.4 M to 0.7 M aceticacid and benzyl alcohol; (c) 0.1 M to 0.5 M acetic acid and 1% benzylalcohol; or (d) 0.5 M to 1.0 M acetic acid and 1% to 2% benzyl alcohol,wherein the contacting step is capable of one or more of a reduction inan amount of spore forming bacteria by at least 3 log₁₀, a reduction inan amount of gram positive bacteria by at least 5 log₁₀, and a reductionin an amount of gram negative bacteria by at least 5 log₁₀ in thechromatography matrix, and the method does not comprise a step ofcontacting the chromatography matrix with a sodium hydroxide solution.5. The method of claim 4, wherein the contacting step is capable of areduction in the amount of one or more of spore forming bacteria, grampositive bacteria, and gram negative bacteria, in the chromatographymatrix, to below the limit of detection as determined by an assayselected from the group consisting of (1) a biofiltration assay, (2)microscopic bacterial staining, (3) IR/FTIR spectroscopy method, (4) asterility test, and (5) a bacterial identification test.
 6. The methodof claim 5, wherein the spore forming bacteria are Bacilluspseudofirmus, the gram positive bacteria are Microbacterium spp., andthe gram negative bacteria are Stenotrophomonas maltophilia.
 7. Themethod of any one of claims 1-2, 3, 4, 5 and 6, wherein the compositionhas pH between 2 and
 3. 8. The method of claim 1, wherein the contactingstep is conducted at a temperature between 15° C. and 30° C.
 9. Themethod of claim 8, wherein the contacting step is conducted at atemperature between 20° C. and 25° C.
 10. The method of claim 1, whereinsaid chromatography matrix comprises a proteinaceous ligand coupled to asupport.
 11. A method for reducing microbial load before applying acomposition comprising a protein for purification of the protein,comprising: providing a chromatography matrix; performing the method ofclaim 1; and applying a composition comprising a protein to thechromatography matrix for purification of the protein.
 12. The method ofclaim 1, wherein the composition consists of 0.4 M to 0.7 M acetic acid.13. The method of claim 1, wherein the composition consists of 0.4 M to0.7 M acetic acid and benzyl alcohol.
 14. The method of claim 1, whereinthe composition consists of 0.4 M to 0.5 M acetic acid.
 15. The methodof claim 1, wherein the composition consists of 0.1 M to 0.5 M aceticacid and 1% benzyl alcohol.
 16. The method of claim 1, wherein thecomposition consists of 0.5 M to 1.0 M acetic acid and 1% to 2% benzylalcohol.
 17. The method of claim 1, wherein the composition consists of:(a) 0.4 M to 0.7 M acetic acid; (b) 0.4 M to 0.7 M acetic acid andbenzyl alcohol; or (c) 0.5 M to 1.0 M acetic acid and 1% to 2% benzylalcohol.
 18. The method of claim 9, wherein the composition consists of:(a) 0.4 M to 0.7 M acetic acid; (b) 0.4 M to 0.7 M acetic acid andbenzyl alcohol; or (c) 0.5 M to 1.0 M acetic acid and 1% to 2% benzylalcohol.